© 2014 Published by The Company of Biologists Ltd | Development (2014) 141, 697-706 doi:10.1242/dev.103978 RESEARCH ARTICLE A nutrient-sensitive restriction point is active during retinal progenitor cell differentiation Nicola K Love1, Nandaki Keshavan1, Rebecca Lewis1, William A Harris1,* and Michalis Agathocleous1,2 KEY WORDS: Differentiation, mTOR, Nutrient deprivation, Proliferation, Restriction point, Retina, Xenopus laevis INTRODUCTION Adequate nutrition is vital for growth (Hietakangas and Cohen, 2009), and nutrient deprivation (ND) has been implicated in disease (Jackson, 2002) and aging (Jasper and Jones, 2010) Despite the fact that growth control is exercised through the precise regulation of stem and progenitor cell lineages, little is known about how this regulation is affected by impaired nutrition Studies in Drosophila show that ND slows cell proliferation (Drummond-Barbosa and Spradling, 2001; McLeod et al., 2010) and that feeding induces quiescent neuroblasts to re-enter the cell cycle (Chell and Brand, 2010; Sousa-Nunes et al., 2011) Although cell cycle control of proliferating populations operates in tandem with the control of differentiation, it is not fully understood how the latter is affected by nutrient availability In Drosophila, ND promotes stem and progenitor cell differentiation in the germline (Hsu and Drummond-Barbosa, 2009) and the lymph gland (Benmimoun et al., 2012; Shim et al., 2012) If stem and progenitor cells deprived of nutrients are forced to differentiate without dividing, tissues could Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK 2Children’s Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA *Author for correspondence (harris@mole.bio.cam.ac.uk) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed Received 19 September 2013; Accepted November 2013 become depleted of stem cells and thus be unable to grow even if food becomes available again However, in most systems it seems that there are mechanisms in place to allow the re-initiation of growth with the return of nutrient availability, suggesting that a particular step in the proliferation-differentiation programme might be sensitive to nutrient loss, so that stem cells are maintained even if other cells are lost To investigate the effects of ND on the proliferationdifferentiation programme, we used amphibian and fish retinas, which grow throughout life by the addition of new cells from the ciliary marginal zone (CMZ), a neurogenic niche at the retina periphery (supplementary material Fig. S1A) (reviewed by Agathocleous and Harris, 2009) Within the CMZ, slowly dividing retinal stem cells reside at the extreme periphery (supplementary material Fig. S1B,C) (Xue and Harris, 2012) Just central to these cells are rapidly proliferating retinal progenitor cells that express markers of neuronal commitment, and at the central edge of the CMZ are cells that are poised to exit the cell cycle and terminally differentiate into neurons and glial cells (Perron et al., 1998; Xue and Harris, 2012) (supplementary material Fig. S1B,C) The CMZ thus recapitulates in space from periphery to centre the temporal sequence of development from uncommitted stem cells, to committed rapidly dividing progenitors, to differentiating neurons (Harris and Perron, 1998) Several studies have elucidated the transcriptional and signalling mechanisms behind this differentiation cascade in the retina (Borday et al., 2012; Cerveny et al., 2010; Denayer et al., 2008; Wehman et al., 2005; El Yakoubi et al., 2012) Here, we examine how nutrient availability regulates the proliferation-differentiation programme in the CMZ We find that ND induces an mTOR-dependent block in proliferation and differentiation specifically in progenitor but not stem cells, defining a nutrient-sensitive restriction point that operates in the differentiation programme of retinal stem and progenitor cells RESULTS Nutrient deprivation causes a decrease in CMZ proliferation We deprived Xenopus laevis larvae of their physiological internal nutrients (Jorgensen, 2008; Jorgensen et al., 2009) by dissection of the yolk deposits in the gut, starting at stage 34/35 (Fig 1A), ~36 hours post-fertilisation (hpf) From this stage onwards, cells in the central retina are mostly postmitotic, with proliferating cells confined to the CMZ (Holt et al., 1988) These ND embryos survived well and days later, at stage 41, were morphologically normal (Fig. 1B,C), although smaller than controls with proportionally smaller eyes (Fig. 1D-F) The CMZs of ND larvae were smaller and had fewer cells than controls (Fig. 1E,F) ND caused a significant drop in incorporation of the nucleotide analogue EdU in the CMZ (Fig. 1G,H) There was no conspicuous increase in CMZ cell apoptosis (supplementary material Fig. S2AD) These results suggest that ND prevents proliferation in the CMZ The loss of proliferation was due to ND rather than injury 697 Development ABSTRACT In many growing tissues, slowly dividing stem cells give rise to rapidly proliferating progenitors that eventually exit the cell cycle and differentiate Growth rates are limited by nutrient availability, but it is unclear which steps of the proliferation-differentiation programme are particularly sensitive to fuel supplies We examined how nutrient deprivation (ND) affects stem and progenitor cells in the ciliary marginal zone (CMZ) of the amphibian retina, a well-characterised neurogenic niche We show that ND specifically blocks the proliferation and differentiation of progenitor cells through an mTORmediated mechanism By contrast, the identity and proliferation of retinal stem cells are insensitive to ND and mTOR inhibition Refeeding starved retinas in vitro rescues both proliferation and differentiation, and activation of mTOR is sufficient to stimulate differentiation even in ND retinas These results suggest that an mTOR-mediated restriction point operates in vivo to couple nutrient abundance to the proliferation and differentiation programme in retinal progenitor cells RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 1 Nutrient deprivation causes a decrease in CMZ proliferation (A) The nutrient deprivation (ND) procedure The yolk-rich gut (yellow area), which feeds the embryo, was removed by dissection (red dashed line), leaving the animal in a nutrientdeprived state Xenopus ND embryos appeared developmentally and morphologically normal days after yolk dissection when compared with control animals (B), but were smaller (C) with smaller eyes (D; control on left, ND embryo on right) (E,F) The cross-sectional area of the whole retina was reduced after ND ND also reduced the size of the ciliary marginal zone (CMZ) (H) and decreased CMZ proliferation as measured by EdU incorporation after a 2-hour pulse (G,H) In G, embryos were nutrient-deprived during the stages shown n=10-20 retinas per condition (I) ND decreases both the domain and intensity of cyclin D1 (J) ND significantly decreased EdU incorporation in progenitor (Hes1 negative) cells but did not affect proliferation in the Hes1-positive stem cell population n=3, 12 embryos/condition (G,J) Error bars show 95% confidence intervals; ***P≤0.001 (unpaired t-test); ns, not significant Scale bars: 100 μm (E,F); 50 μm (H,I) 698 The reduction in CMZ proliferation is due to a specific effect on retinal progenitor cells To test whether all CMZ cells are similarly impaired in proliferation by nutrient withdrawal, or whether stem and progenitor cells respond differently, we measured EdU incorporation in cells positive for the stem cell marker Hes1 (M Perron, personal communication) We saw a significant decline in the proliferation of EdU-positive progenitors in the Hes1-negative compartment Surprisingly, however, there was no reduction in the number of EdU/Hes1 double-positive cells (Fig. 1J) This argues that progenitor but not stem cell proliferation is particularly sensitive to nutrient abundance Nutrients are necessary for the acquisition of the committed neural progenitor fate but not for the maintenance of the most immature stem/progenitor cells We next investigated whether the reduced size and cell number of the CMZ after ND in the absence of cell death is solely due to changes in proliferation or whether impairments in the Development to the embryo, as no reduction in proliferation was observed when less than a quarter of the yolk was removed (supplementary material Fig. S3) To test whether the effects of ND on the CMZ were specific to Xenopus, we also deprived zebrafish larvae of nutrients by removing the majority of their yolk at 56 hpf Both the number of CMZ cells (supplementary material Fig. S4A) and CMZ proliferation (supplementary material Fig. S4B,C) were decreased in ND fish at 84 and 112 hpf CMZ proliferation is therefore dependent on abundant nutrients in both fish and frogs Detection of cell cycle gene expression by in situ hybridisation showed that ND inhibited the expression levels of cyclin A2 and cyclin D1 ND did not significantly change the expression levels of the cyclin-dependent kinase inhibitor p27Xic1, although it appeared to expand its expression domain (Fig. 1I; supplementary material Fig. S5A) These data indicate that the reduction in CMZ proliferation after ND might be ultimately mediated by changes in the expression of specific cell cycle regulators RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 2 Nutrients are necessary for acquisition of the committed neural progenitor fate but not for the maintenance of the most immature stem/progenitor cells (A) Proneural gene expression levels decreased following ND, as did levels of the progenitor markers sox2 and n-Myc (B) Dashed line in B indicates the central domain of the CMZ n=8, 32 embryos/condition (C) Expression of the eye field transcription factor gene rx1 remained unchanged n=4, 16 embryos/condition (C) c-Myc gene expression levels remained unchanged; however, the domain of c-Myc gene expression was reduced in the central CMZ of ND embryos n=8, 32 embryos/condition (D) Expression levels of the stem cell marker genes hes1 and hes2 remained unchanged in ND conditions n=4, 16 embryos/condition Scale bars: 50 μm (A,B,D); 50 μm (C; rx1) 25 μm (C; c-Myc) contracting in the central CMZ (Fig. 2C, Fig. 6D) The very peripheral cells of the CMZ additionally express the hairy and enhancer-of-split (hes) genes hes1 and hes4, which are believed to be markers of retinal stem cells (Parain et al., 2012; El Yakoubi et al., 2012) Following ND, expression of hes1 and hes4 remained high in the peripheral CMZ (Fig. 2D, Fig. 6E) These results suggest that, unlike the progenitors, the stem cells in the extreme periphery of the CMZ are relatively immune to ND Committed progenitor cells continue to differentiate normally following ND Retinal progenitors in the more central CMZ express proneural genes, committing them to differentiation (Harris and Perron, 1998; Perron et al., 1998) In ND retinas, these progenitors are lost, indicating that their production ceases But what happens to the progenitors that were already present at the start of ND? Do they complete their differentiation programme even in the absence of continued proliferation? To answer this question, embryos were exposed to EdU for just 20 minutes, and then washed and nutrient deprived The fate of these EdU-positive cells was examined days later As would be expected in normal retinas, we found EdU cells located in the differentiated layers of the retina (Fig 3A) However, and perhaps surprisingly, ND retinas showed significantly more EdU-positive terminally differentiated cells outside of the CMZ than normal embryos, suggesting that many progenitors quickly exited the cell cycle and differentiated after ND (Fig. 3B) Some of these EdU-positive cells were labelled with Islet1, a marker of terminal differentiation of 699 Development differentiation programme are also involved Early stage progenitor cells express n-Myc (Xue and Harris, 2012), Sox2 (Agathocleous et al., 2009; Van Raay et al., 2005) and several proneural basic helixloop-helix (bHLH) genes (Perron et al., 1998) These transcription factors define the progenitor fate and are necessary for neurogenic competence We assayed the CMZ for their expression by in situ hybridisation The domain of expression of these progenitor genes was reduced, suggesting fewer progenitors, in agreement with our data for a smaller CMZ and specific deficits in progenitor proliferation (Fig 2A,B; supplementary material Fig. S5B), Crucially, however, we also found that the levels of expression of the bHLH genes ascl1, atoh7, neurogenin2, neuroD and ath3 and of sox2 and n-Myc were all strongly reduced by ND, as judged by the intensity of in situ hybridisation staining (Fig. 2A,B; supplementary material Fig. S5B; see Fig. 6A,B for quantification) As these genes are crucial for differentiation, these findings suggest that ND not only depletes the CMZ of progenitors, but also blocks the progenitor differentiation programme upstream of proneural genes We then examined the most peripheral population, where retinal stem cells reside The eye field transcription factor gene rx1 is expressed throughout the CMZ, including the extreme peripheral population (Perron et al., 1998) Its expression was unaltered following ND (Fig. 2C, Fig. 6C) The retinal stem cells at the extreme periphery express the transcription factor c-Myc in the absence of n-Myc, and this gene is also expressed along with n-Myc in the more central CMZ where progenitor cells reside (Xue and Harris, 2012) c-Myc was still strongly expressed in the most peripheral domain after ND, despite its domain of expression RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 CMZ cells are not lost and are still responsive to growth-promoting conditions Decreasing nutrient levels from 60% to 30% L15 for a 2hour period decreased CMZ proliferation acutely in a dose-dependent manner (Fig. 4C), suggesting that proliferation is dynamically, and rapidly, attenuated by nutrient availability We next asked how quickly re-feeding initiates the repopulation of the CMZ Remarkably, in as little as 20 minutes after explanting eyes into L15, a burst of EdU uptake could be observed in the peripheral edge of the CMZ (Fig. 4D), and by 2 hours there was a large increase in EdU uptake in the CMZ compared with ND retinas (Fig. 4E) After 24 hours, re-fed retinas showed increased expression of the cell cycle gene cyclin D1 and a significant upregulation of the proneural genes ascl1 and atoh7 (Fig. 4F,G; supplementary material Fig. S6A) These results demonstrate that the retinal stem cells at the CMZ periphery, and the progenitors whose proliferation had been blocked by ND, are still functional and, with a new supply of nutrients, are able to reinitiate proliferation and differentiation to reconstitute the entire CMZ Fig. 3 Committed progenitor cells continue to differentiate normally following ND (A) Many cells labelled with EdU 20 minutes prior to ND were found in the differentiated central retina after a 3-day chase period n=3, 12 embryos/condition (B) These cells increased in number in the ND condition compared with control retinas n=3, 12 embryos/condition Error bars show 95% confidence intervals; *P≤0.05 (unpaired t-test) (C,D) High magnification of the central retina, which contains differentiated cells Some EdU-labelled cells were also positive for the neuronal marker Islet1, which marks specific subsets of amacrine and bipolar cells in the inner nuclear layer of the differentiated retina n=3, 12 embryos/condition Scale bars: 40 μm (C); 10 μm (D) several retinal cell types in Xenopus [e.g ON-bipolar, cholinergic amacrine and ganglion cells (Elshatory et al., 2007)] (Fig. 3C,D), suggesting that these cells not only had left the CMZ, but also had completed their differentiation programme This experiment, in combination with our previous results, suggests that the stem and progenitor cells of the CMZ respond differently to ND Stem cells maintain their undifferentiated state, whereas many committed progenitors differentiate The production of new progenitors is blocked because the expression of genes that direct the progenitor fate is also inhibited This depletes the CMZ of progenitor cells while maintaining the stem cells at the periphery of the CMZ Re-feeding promotes reconstitution of the CMZ If retinal stem cells are maintained during ND, then feeding a starved embryo may repopulate the CMZ Unfortunately, ND by dissection of yolk prevents feeding in vivo, as the experimental animals lack intestinal tracts To circumvent this problem, we explanted whole eyes from ND animals into nutrient-rich media We found that culturing retinas from ND embryos ex vivo in L15 medium, containing nutrients but no serum or growth factors, resulted in a significant increase in CMZ proliferation (Fig 4A,B), demonstrating functionally that ND 700 The serine/threonine kinase mTOR regulates cell growth, proliferation and survival by sensing and responding to nutrient availability (Sengupta et al., 2010; Wang and Proud, 2009) and is involved in the regulation of Drosophila eye development (Lanet et al., 2013; McNeill et al., 2008) To test whether mTOR regulates the effects of ND on the CMZ, we first immunostained for phosphoRibosomal protein S6 (pS6), the cardinal marker of mTOR activity pS6 was mostly absent from the differentiated cells in the retina but stained the CMZ intensely (Fig 5A) Within the CMZ, it marked the progenitor cells, but not the most peripheral undifferentiated stem cells (Fig. 5B) pS6 staining was reduced by ND to levels similar to those of embryos treated with the mTOR inhibitor rapamycin (Fig. 5A,B) Thus, in CMZ progenitors, as in other systems, nutrients are necessary for mTOR activity We next asked whether mTOR is necessary for CMZ proliferation Incubating embryos with rapamycin, like ND, decreased eye growth (Fig. 5C) and inhibited proliferation in the CMZ (Fig. 5D) Both rapamycin treatment and ND reduced the fraction of the G2/M population that was EdU positive after a 1.5-hour pulse, suggesting a G2/M slowdown (Fig. 5E,F) Also similarly to ND, rapamycin inhibited expression of Cyclin D1 and Cyclin A2 (Fig. 5G), without significantly changing the level of p27Xic1 expression (Fig. 5G) These results suggest that mTOR signalling is sensitive to nutrient levels and is necessary for cell proliferation in the CMZ Rapamycin caused changes in gene expression similar to those observed with ND The intensity of expression of the proneural genes ascl1, atoh7, ath3 and neurogenin2 in the central CMZ was significantly decreased both by ND and rapamycin treatment (Fig 6A) Rapamycin also reduced the expression levels of the central progenitor markers sox2 and n-Myc, which lie upstream of proneural genes in the differentiation cascade (Van Raay et al., 2005; Xue and Harris, 2012) (Fig. 6B), but did not attenuate the expression of rx1 (Fig. 6C), c-Myc (Fig. 6D), hes1 or hes4 (Fig. 6E), as expressed by the most peripheral CMZ stem cells These results show that blocking mTOR activity mimics many of the effects of ND in the CMZ, raising the possibility that mTOR mediates the effects of ND This hypothesis predicts that stimulation of the mTOR pathway in the absence of nutrients might rescue the ND phenotype mTOR can be activated by the insulin mimetic compound and PTEN inhibitor bpV(phen) (hereafter bpV), which promotes PI3K signalling (Posner et al., 1994; Schmid et al., 2004) Development The mTOR pathway is involved in the nutrient-sensitive restriction point RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 When we treated ND larvae with bpV, we found a robust rescue of pS6 specifically in the CMZ (Fig 7A,B), similar to what was observed in explant cultures upon re-feeding (supplementary material Fig. S6B) Activation of mTOR by bpV in ND embryos was also sufficient to rescue n-Myc and sox2 expression in the central CMZ (Fig. 7C,F) and the expression of the proneural genes ascl1 and atoh7 (Fig. 7D,F) Interestingly, bpV did not cause an upregulation of pS6 in the CMZ stem cell zone at the far periphery, which also maintained expression of hes1 and hes4 (Fig. 7E), consistent with the restoration of mTOR signalling only in its physiological spatial domain Treatment of control embryos with bpV did not result in an upregulation of pS6, nor did it have an influence on progenitor proliferation or differentiation (supplementary material Fig S7A-E) 701 Development Fig. 4 Re-feeding promotes reconstitution of the CMZ (A,B) Explanted retinas from ND embryos grown in L15 medium containing sugars and amino acids for 24 hours showed an increase in CMZ proliferation after a 2-hour pulse with EdU n=3, 18 retinas/condition (C) This increase in proliferation varied with nutrient levels, suggesting that nutrients can regulate proliferation within a dynamic range n=3, 18 retinas/condition (D,E) Proliferation was initiated in the CMZ as early as 20 minutes after re-feeding (D), with robust proliferation being observed 2 hours after the addition of nutrients (E) (F,G) After culture for 24 hours in L15, a strong upregulation of Cyclin D1 and the proneural genes ascl1 and atoh7 was observed in re-fed retinas n=3, 18 retinas/condition (B,C,G) Error bars show 95% confidence intervals; ***P≤0.001 (unpaired t-test) Scale bars: 50 μm (A,D,E,F) RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 5 The mTOR pathway is involved in the nutrient-sensitive restriction point (A) The CMZ stained strongly for the mTOR target pS6 (red) in control embryos, whereas pS6 staining was lost in the CMZ of ND embryos or in embryos treated with the mTOR inhibitor rapamycin (n=40/40 embryos) (B) Within the CMZ, pS6 was present in committed progenitors but not in undifferentiated stem/progenitor cells (n=40/40 embryos) (C) Rapamycin or ND resulted in a significantly reduced eye size in stage 41 embryos n=3, 12 embryos/condition (D) Rapamycin treatment during stages 38-45 significantly reduced the number of proliferative cells in the CMZ n=5-12 retinas (E,F) ND or rapamycin reduced the fraction of G2/M cells that were EdU positive after a short EdU pulse (E), suggesting G2/M slowdown, and reduced the overall percentage of EdUpositive cells in the retina (F) from 13% in controls to 9% in rapamycin-treated and 7% in ND embryos n=3 experiments (G) Expression levels for the cell cycle genes cyclin D1, cyclin A2 and p27Xic1 ND and rapamycin expression levels are shown as ratio to control n=6, 24 embryos/condition (C-E,G) Error bars show 95% confidence intervals; ***P≤0.001, *P≤0.05 (unpaired t-test) Scale bars: 100 μm (A); 50 μm (B) 702 are mediated by mTOR, its effects on proliferation are likely to be mediated by additional cellular mechanisms These results support the idea that mTOR signalling, which is lost as a result of ND, is essential for both the differentiation and proliferation of retinal progenitor cells Even in the absence of nutrients, mTOR is sufficient to drive the progenitor differentiation programme Retinal stem cells, by contrast, are not dependent on mTOR or abundant nutrients for their maintenance and proliferation DISCUSSION We introduce a new ND paradigm for frog and fish embryos and use it to uncover a nutrient-sensitive restriction point in the proliferationdifferentiation programme in the CMZ, a well-organised retinal stem cell niche When embryos are deprived of nutrients, proliferation in the CMZ is shut down and committed progenitors quickly Development Although bpV rescued the differentiation block after ND, it failed to rescue the decrease in eye size (Fig 8A) and proliferation (Fig. 8B) caused by ND Consequently, the block by ND on progenitor differentiation can be uncoupled from, and is not simply downstream of, the impairment in progenitor proliferation It is interesting to note in this regard that retinas from ND animals show a transient increase in EdU incorporation 2 hours after the addition of bpV (Fig. 8D,E), and a partial rescue of Cyclin D1 expression (Fig. 8C) One possibility is that mTOR activation in the absence of nutrients does attempt to turn on the proliferation programme but, despite mTOR being necessary for progenitor proliferation (Fig. 5), it is insufficient to drive proliferation in the absence of actual nutrients Indeed, at 24 hours of bpV treatment, proliferation receded to the levels observed in ND embryos (Fig. 8D,E) Therefore, whereas the effects of ND on differentiation RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 6 Activation of mTOR by nutrients is necessary for neural commitment and differentiation but not for the maintenance of the most immature stem/progenitor cells (A,B) Proneural gene expression levels were decreased in ND and rapamycin-treated embryos (A), as were levels of the progenitor markers sox2 and n-Myc (B) n=8, 32 embryos/condition (C) Expression of the eye field transcription factor gene rx-1 remained unchanged by ND n=4, 16 embryos/condition (D) c-Myc expression levels remained unchanged; however, the domain of c-Myc expression was reduced in the central CMZ of ND and rapamycin-treated embryos n=8, 32 embryos/condition) (E) Expression levels of the stem cell marker genes hes1 and hes4 remained unchanged (n=4, 16 embryos/condition In all cases, error bars show 95% confidence intervals; ***P≤0.001, **P≤0.01, *P≤0.05 (unpaired t-test) reflect a strategy used by tissues to limit stem cell differentiation when nutrients are scarce and to renew growth by stimulating proliferation of rapidly dividing progenitors when nutrients become available How is it that stem cells, unlike progenitors, are relatively immune to ND? It is possible that they express negative mTOR regulators such as TSC2, which limits mTOR signalling in Drosophila intestinal stem cells but not progenitors (Kapuria et al., 2012), or that stem cells are more adept than progenitors at using low levels of nutrients for proliferation, perhaps because of their slower cycling (Xue and Harris, 2012) In summary, our results support a model in which abundant nutrients activate mTOR causing both proliferation and production of committed retinal progenitor cells When mTOR is inhibited under conditions of ND, these cells differentiate quickly but are not replenished The remaining, presumably earlier stage, progenitors stop dividing and their differentiation programme is blocked upstream of the proneural genes Thus, ND leads to the preferential loss of committed progenitors, but it does not block retinal stem cell fate or proliferation Our results thus define a nutrient-sensitive restriction point in the proliferation-differentiation programme within retinal progenitor cells in the CMZ, and provide a mechanism whereby growth can be restricted, when nutrients are unavailable, without losing the capacity to reinitiate growth if conditions of plenty return MATERIALS AND METHODS Animal maintenance and pharmacological treatment Xenopus laevis embryos, obtained by fertilisation, were raised in 0.1× modified Barth’s solution (MBS) and staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967) Larvae were nutrient deprived at stage 34/35 and analysed at stage 41 unless otherwise indicated For drug treatments, 1-5 μM rapamycin (Calbiochem) or 300 nM bpV(phen) (Calbiochem) were bath applied in embryo medium at stages 28 and 34, respectively, or at stages otherwise indicated Zebrafish embryos were raised at 25-32°C and staged as hpf (Kimmel et al., 1995) All animal work was approved by Local Ethical Review Committee at the University of Cambridge and performed in accordance with the UK Home Office license PPL 80/2606 703 Development differentiate However, the slowly dividing stem cells at the extreme periphery of the CMZ are maintained throughout ND Our results indicate that nutrient abundance controls the expression of multiple genes required for a specific step in the proliferation-differentiation programme, defining a restriction point downstream of retinal stem cells This means that the CMZ can be repopulated and growth can re-initiate as soon as nutrients become available again Previous work in adult flies and mice showed that some intestinal and germline stem cells are maintained during caloric restriction or amino acid deprivation, whereas differentiated progenitor populations may be more reduced (Drummond-Barbosa and Spradling, 2001; McLeod et al., 2010; Yilmaz et al., 2012) Although differences are expected between the physiology of adult caloric restriction and embryonic ND, the nutrient-sensitive restriction point that we describe here could play a role in other tissues to enable stem cells to preferentially withstand ND compared with restricted progenitors Key to the response of progenitor cells to nutrient availability is the cellular nutrient-sensing pathway mediated by mTOR Several growth factors, including the mTOR activator insulin/IGF, promote retinal progenitor proliferation (Agathocleous and Harris, 2009) Activation of mTOR by deletion of PTEN, a negative regulator of the PI3K/mTOR pathway, leads to premature neurogenesis in the developing central retina (Jo et al., 2012), suggesting that the PI3K/mTOR pathway regulates differentiation Similarly, TOR signalling promotes differentiation in the Drosophila eye (Bateman and McNeill, 2004) and the chick neural tube (Fishwick et al., 2010) The mTOR pathway has been implicated in antagonising stem cell self-renewal in several systems mTOR activation can deplete stem cells in mouse haematopoietic stem cells (Yilmaz et al., 2006; Chen et al., 2008), Drosophila germ stem cells (Sun et al., 2010) and mouse hair follicle stem cells (Castilho et al., 2009) In the subventricular zone (SVZ), mTOR is required for the proliferation of transit-amplifying progenitors, but is absent from SVZ populations that include neural stem cells (Paliouras et al., 2012) Our results suggest that this general function of mTOR in promoting committed progenitor fates in a variety of tissues might RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 7 Activation of mTOR signalling is sufficient to maintain the progenitor pool (A,B) Activation of mTOR with bpV rescues pS6 to a level 70% of that of controls in ND retinas (top row), specifically in CMZ progenitor cells (bottom row) Dashed lines encircle the CMZ n=4, 16 embryos/condition (C-F) bpV treatment rescued the downregulation of genes observed following ND, without affecting the expression of stem cell markers n=4, 16 embryos/ condition (B-E) Error bars show 95% confidence intervals; ***P≤0.001, *P≤0.05 (unpaired t-test) Scale bars: 100 μm (A, top panel); 50 μm (A, bottom panel); 50 μm (F) 704 Immunostaining Immunostaining was performed on 12 μm sections using rabbit anti-active Caspase (1:500; BD Biosciences; 59565), rabbit anti-phospho-Ribosomal protein S6 (Ser235; 1:500; Upstate; 07-433), mouse anti-Islet1 (1:100; Developmental Studies Hybridoma Bank; 39.3f7), Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-rabbit (both 1:1000; Invitrogen) Antigen retrieval was performed by steaming with 0.01 M sodium citrate (pH 6) prior to staining for pS6 Nuclei were labelled with 0.1 μg/ml DAPI (Invitrogen) Immunofluorescent sections were imaged with an Olympus Fluoview FV1000 laser-scanning confocal microscope or with a Nikon Development Dissection of yolk from Xenopus and zebrafish Xenopus and zebrafish embryos, like other externally developing organisms, rely on maternally derived yolk platelets for nutrition during the first few days of life (Jorgensen et al., 2009) The yolk sacs of stage 34 Xenopus embryos were removed by microdissection using fine dissecting pins as illustrated (Fig. 1A) Removal of yolk from larval zebrafish was carried out at 56 hpf in methylcellulose by first making a fine lesion with a dissection pin, and then carefully removing the contents of the yolk sac by suction through a glass pipette In both Xenopus and zebrafish care was taken to avoid damaging the blood vessels and maintain blood circulation RESEARCH ARTICLE Development (2014) doi:10.1242/dev.103978 Fig. 8 Activation of mTOR signalling is sufficient to initiate, but not maintain, progenitor proliferation (A-C) Eye size at stage 41 was not rescued by bpV (A), nor was proliferation (B), despite expression of Cyclin D1 being significantly higher than in ND embryos (C) n=4, 16 embryos/condition Error bars show 95% confidence intervals; *P≤0.05 (unpaired t-test) (D,E) In explanted cultures a short-term incubation (2 hours) with bpV was sufficient to initiate proliferation in ND retinas; however, bpV treatment was not sufficient to maintain proliferation for longer periods n=4, 20 retinas/condition Scale bars: 50 μm (E,F) BrdU/EdU labelling To mark cycling cells, mM EdU was bath applied to Xenopus embryos, or embryos were injected with BrdU or EdU hours prior to fixation For pulse chase experiments, mM EdU was bath applied with 1% DMSO for 20 minutes before being washed out BrdU incorporation was detected as previously described (Agathocleous et al., 2009), and EdU detection was by Click-iT chemistry performed in accordance with the manufacturer’s instructions (Invitrogen) Flow cytometry was performed using a CyAN flow cytometer (Beckman Coulter) and analysed using FlowJo software (Tree Star) Statistical analysis was determined by Student’s t-test or oneway ANOVA In situ hybridisation In situ hybridisation was performed on 20 μm sections as described previously (Xue and Harris, 2012) using digoxigenin-labelled antisense mRNA probes against rx1 (Mathers et al., 1997), atoh7 (Kanekar et al., 1997), ascl1, neurogenin2 (a gift from Dr Anna Philpott, University of Cambridge, UK), ath3 (Takebayashi et al., 1997), c-Myc and n-Myc (Bellmeyer et al., 2003), sox2 (Mizuseki et al., 1998), p27Xic1, cyclin A2 (Howe et al., 1995), cyclin D1 (a gift from Dr Tim Hunt, CRUK, UK) and hes1 and hes4 (a gift from Dr Muriel Perron, Universite Paris-Sud, France) Images were acquired using a Zeiss Axioskop microscope and Q-imaging camera and staining intensity was analysed using ImageJ Intensity measurements were normalised to background intensity, then the intensity ratio of experimental conditions relative to controls was determined Statistical significance was tested using a Student’s t-test Explant cultures Stage 41 Xenopus embryos were deprived of their yolk for 24 hours prior to retinal dissection For ex vivo re-feeding conditions, explanted retinas from control and nutrient-deprived embryos were cultured in a 60% solution of Leibovitz L-15 medium (Sigma) containing galactose and amino acids as nutrient sources For ex vivo ND, retinas were cultured in 1× MBS, a salt buffer without nutrients In proliferation experiments, explants were cultured for 22 hours at 20°C then incubated with EdU for 2 hours prior to fixation Explants for in situ hybridisation were cultured in either 60% L15 or 1× MBS for 24 hours and then fixed The insulin mimetic bpV was used to activate mTOR in explanted retinas from embryos deprived of nutrients for 24 hours Eyes were cultured in 300 nM bpV for or 24 hours To label proliferating cells, explants were either cultured at 20°C with EdU for hours, or cultured for 22 hours and EdU added for hours prior to fixation Acknowledgements We thank N Coutts for technical assistance and N Miller for help with flow cytometry We are also grateful to A McNabb, K Scott, T Dyl and S.Dudczig for frog and fish maintainance Competing interests The authors declare no competing financial interests Author contributions N.K.L performed most of the experiments and co-wrote the paper with M.A M.A initiated the project and performed experiments in Fig 1A-H and Fig 5A-F N.K generated the data in supplementary material Fig S4 and R.L generated data in supplementary material Fig S3 W.A.H guided the research and helped write the manuscript Funding N.K.L is a member of the Wellcome Trust 4-Year Programme in Stem Cell Biology W.A.H is funded by a Senior Investigator Award (SIA) grant from the Wellcome Trust M.A was funded by a Gonville and Caius College Research Fellowship and an 1851 Royal Commission Research Fellowship Deposited in PMC for immediate release Supplementary material Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.103978/-/DC1 References Agathocleous, M and Harris, W A (2009) From progenitors to differentiated cells in the vertebrate retina Annu Rev Cell Dev Biol 25, 45-69 Agathocleous, M., Iordanova, I., Willardsen, M I., Xue, X Y., Vetter, M L., Harris, W A and Moore, K B (2009) A directional 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suppression of TSC2 expression regulates cell differentiation in the Drosophila intestinal stem cell lineage PLoS Genet 8, e1003045 Development (2014) doi:10.1242/dev.103978 706 ... re-initiate as soon as nutrients become available again Previous work in adult flies and mice showed that some intestinal and germline stem cells are maintained during caloric restriction or amino acid... Holland Publishing Company: Amsterdam, The Netherlands Paliouras, G N., Hamilton, L K., Aumont, A. , Joppé, S E., Barnabé-Heider, F and Fernandes, K J L (2012) Mammalian target of rapamycin signaling... 2461-2469 Takebayashi, K., Takahashi, S., Yokota, C., Tsuda, H., Nakanishi, S., Asashima, M and Kageyama, R (1997) Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix